The use of nuclear transfer technology to produce animals in conjunction with the development of robust sources of identical donor nuclei has the potential for creating transgenic and/or genetically-altered animals using gene manipulation technology, such as gene targeting or site-directed mutagenesis. In addition, the production of genetically identical animals is of potential importance to biomedical research by eliminating genotypic variation between animals receiving different experimental manipulations to allow greater statistical validity with fewer animals. The PI has established a successful in vitro fertilization (IVF) program at the ORPRC where rhesus monkeys have been produced for the first time by nuclear transfer from embryonic cells. The technology employs enucleated, metaphase II oocytes as recipient cytoplasts and individual blastomeres from IVF-produced embryos as nucleus donors. The proposed work is designed to improve the nuclear transfer procedures in rhesus macaques with the goal of making genetically identical animals available to the biomedical community for experimental use, while evaluating the efficiency and cost-effectiveness of this evolving approach. The three Specific Aims are: 1) to optimize the preparation, maturation, activation, and low temperature storage of MII cytoplasts. Conditions for the cryopreservation of MII cytoplasts will be established and in vitro maturation conditions will be sought to take advantage of the potentially large numbers of ovarian oocytes; 2) to establish a source of totipotent embryonic cells or cultured ES cells as nuclear donors. Stem cell lines will be created from in vivo produced blastocysts and stably transfected with a molecular marker, such as green fluorescent protein (GFP), in collaboration with Dr. James Thomson of the WiRPRC. The totipotency of these lines will be evaluated in reconstituted embryos following nuclear transfer. Additionally, inner cell mass cells from IVF-produced embryos will be evaluated as nuclear donors and as a source of ES cell lines; and 3) to produce rhesus monkeys by conventional or serial nuclear transfer, and evaluate blastomere separation and culture as an alternative method of cloning. The applicants propose to produce five sets of genetically identical animals in the initial year, and more thereafter by nuclear transfer. Also, the simplicity of separating IVF-produced embryos at early cleavage stages with the transfer of individual blastomeres or groups of blastomeres to surrogate zonae offers a low technology method of cloning that will be evaluated.